Enzymes utilize organic radicals to catalyze a variety of important metabolic reactions. The overall goal of this research is to delineate the mechanistic details of radical generation and control by these enzymes. This research focuses on three radical enzymes: benzylsuccinate synthase (BSS) and its activating enzyme (AE) in the K99 phase, and spore photoproduct lyase (SPL) in the ROD phase. The later two enzymes belong to the radical SAM superfamily, which utilize S-adenosylmethionine (SAM) coupled by a unique [4Fe-4S] cluster to generate the reactive organic radicals. BSS, found in bacteria like T. Aromatica, converts toluene to benzylsuccinate by a mechanism involving protein-based glycyl radical to initiate the toluene anaerobic biodegradation process. This research is of significance due to the fact that toluene and its related compounds comprise a major category of pollutants with neuronal and cancer-promoting toxicity. To generate the glycyl radical, an activating enzyme (AE) is needed, which oxidizes the glycine residue using a S'-deoxyadenosyl radical generated, in turn by reductive cleavage of SAM with the electron provided by a [4Fe-4S] cluster. To investigate this BSS-catalyzed toluene bioremediation process, biochemical, spectroscopic, and mutagenic studies of both BSS and BSS-AE will be pursued. The specific aims include the investigations of enzyme kinetics, the functions of different [4Fe-4S] clusters in BSS/BSS-AE, and the roles of different organic radical intermediates during the catalysis. SPL exists in the spores of bacteria such as B. subtilis, and repairs unique T-T crosslink 5-thyminyl-5, 6- dihydrothymine (commonly called spore photoproduct, SP) formed upon UV irradiation. SPL adopts a "direct reverse" strategy to repair SP, which involves neither removal nor replacement of bases or nucleotides. It thus represents a unique DNA repair pathway in Nature. To understand the SPL mediated DNA repair reaction, chemical, kinetic, spectroscopic, and mutagenic methods will be employed. The objectives include: investigating the SP repair in a wide range of DNA secondary structures, probing the reaction mechanism by SP analogues (mechanism-based enzyme inhibitors), and examining the kinetic isotope effects and reaction reversibility. In addition, the redox potential of the [4Fe-4S] cluster will be determined and the influence of SAM and key amino acids to the redox potential will be investigated.